Nonfat dry milk

These were performed in the muscle biopsy samples obtained from participants in the study. Briefly, total protein was isolated from the muscle biopsy tissue using ice-cold 1X Cell Signaling lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 1 µg/mL leupeptin) and 1 mM PMSF. Protein concentration was determined by Pierce^TM BCA Protein Assay Kit (Thermo Scientific). The proteins were separated by SDS-PAGE followed by electrotransfer on the polyvinylidene difluoride membrane. The membranes were blocked with 5% w/v nonfat dry milk (Cell signaling technology) and probed using antibodies against PGC1α, PPARα, SIRT3, LC3A/B, PINK1, and β-actin, followed by a horseradish peroxidase-conjugated secondary antibody (cell signaling technology). Bands were visualized using the SuperSignal^TM West Dura Extended Duration Substrate (Thermo Scientific) on autoradiography film. Quantification of the immunoblot band intensity was performed using grayscale measurements with ImageJ 1.51j8 software. The expression of proteins of interest were normalized with respective β-actin.

Total protein was extracted from DLX5 knockdown in BM-MSCs and nontargeting siRNA control wells in BM-MSCs using 1× RIPA Buffer containing 1mM PMSF (Cell Signaling Technology, Danvers, MA, USA). Cells were washed with sterile cold PBS and later the lysis extract was centrifuged for 10 min at 4 °C. The concentration of total protein supernatant was determined using the Pierce BCA Protein Assay kit Thermo Fisher Scientific, Waltham, MA, USA. A total of 40 ug protein was resolved on 4–15% Mini-PROTEAN^® TGX™ Precast Gels (Bio-Rad). Blots were transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA). Blocking of the membrane was done at room temperature for 2 h with 5% Nonfat Dry Milk (Cell Signaling Technology, Danvers, MA, USA) in tris-buffered saline with Tween-20 (0.1%, TBST). Membranes were incubated overnight at 4 °C with specific primary antibodies: 1:200 DLX5 (Cat. No.: ab109737; Abcam), 1:100 Caspase-3 (Cat. No.: 9668S; Cell Signaling Technology) and 1:1000 β-Actin (Cat. No.: 8H10D10; Cell Signaling Technology). Later, membranes were incubated with secondary antibodies: IRDye 680RD Goat anti-Rabbit, IRDye 680RD Goat anti-Mouse, and IRDye 800CW Goat anti-Mouse (LI-COR Biosciences, Lincoln, NE, USA) respectively at 1:5000 for 2 h at room temperature. Membranes were imaged using an Odyssey fluorescence scanner (LI-COR Biosciences, Lincoln, NE, USA).

Snap frozen liver samples were homogenized in RIPA buffer with fresh proteinase and phosphatase inhibitor. The concentration of the protein was determined by the bicinchoninic acid assay. Protein sample was prepared with loading buffer (Bio-Rad, 1,610,737) with 5% 2-Mercaptoethanol (Bio-Rad, 161-0710) and subjected to electrophoresis. Protein sample was separated on pre-cast 7.5% or 4-20% polyacrylamide gels (Bio-Rad) and transferred to the PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were stained with Ponceau-S and blocked for 30 min with 5% nonfat dry milk (Cell signaling, 9999) or 5% BSA in Blotto buffer (0.15M NaCl, 0.02M Tris pH 7.5, 0.1% Tween in dH2O), and incubated with primary antibodies at 4°C overnight at the following concentrations: GS (Sigma, G2781, 1:2000), CYP2E1 (Sigma, HPA009128, 1:1000), CYP1A2 (Santa Cruz Biotechnology, sc-53241, 1:1000), Cyclin D1 (Abcam, ab134175, 1:1000). Membranes were washed in Blotto buffer and incubated with the appropriate HRP-conjugated secondary antibody for 60 min at room temperature. Membranes were washed with Blotto buffer, and bands were developed utilizing SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, 34,080) and visualized by time-gradient autoradiography.